Simultaneous expression of germline gamma1 and epsilon immunoglobulin heavy chain transcripts in single murine splenic B-cells.
While successive isotype switching can occur from IgM to IgE via IgG1, little is known about the pattern of germline transcript expression in normal B cells committed to switching to multiple isotypes. In this study we define the relationship between germline transcript expression and immunoglobulin isotype expression and secretion in murine splenic B cells. Following 7 days stimulation with LPS and IL-4, 10% of single cells secrete IgE and of these only 1 in 10 secrete IgE alone. In contrast, when cells were stimulated with LPS and IL-4 for 48 hr prior to sorting into clonal cultures, 71% secreted IgE alone. In an attempt to isolate switch intermediates, IgG1+IgM- cells were sorted and upon re-culture secreted predominantly IgG1 alone or IgG1 and IgE. The frequency of cells expressing germline epsilon transcripts was 34% for surface IgG1+IgM- expressing cells and 14% for surface IgG1-IgM+ negative cells. Furthermore, analysis of single surface IgG1+IgM- cells demonstrated that these cells can co-express germline gamma1 and epsilon transcripts. Sorting of IgG1-IgM+ cells according to surface expression of the integral membrane proteoglycan, Syndecan, demonstrated that IgM+Syndecan+ cells had a lower frequency of germline transcript expression and a lower frequency of isotype switching (32%) compared to IgM+Syndecan cells (83%). Collectively, these finding show that murine B cells can switch successively from IgM to IgE via IgG1 and that IgG1 expressing intermediates express germline transcripts. Furthermore, Syndecan expression appears to be linked to germline CH transcription and isotype switch commitment.
Goodman
D J
DJ
The Walter and Eliza Hall Institute of Medical Research, The Royal Melbourne Hospital, Parkville, Victoria, Australia. goodman@svhm.org.au
Gerondakis
S
S
D000818:Animals; D001402:B-Lymphocytes / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D002999:Clone Cells; D018095:Germ-Line Mutation; D017578:Immunoglobulin Class Switching / Q000187:drug effects; D007073:Immunoglobulin E / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000276:immunology; D007075:Immunoglobulin M / Q000276:immunology; D015847:Interleukin-4 / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000096:biosynthesis; D051379:Mice; D008807:Mice, Inbred BALB C; D011509:Proteoglycans / Q000096:biosynthesis; D013154:Spleen / Q000166:cytology*; D053667:Syndecans; D014158:Transcription, Genetic
D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007075:Immunoglobulin M; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D011509:Proteoglycans; D053667:Syndecans; D015847:Interleukin-4; D007073:Immunoglobulin E